The human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression.

BACKGROUND: The accurate alignment of chromosomes at the spindle equator is fundamental for the equal distribution of the genome in mitosis and thus for the genetic integrity of eukaryotes. Although it is well established that chromosome movements are coupled to microtubule dynamics, the underlying mechanism is not well understood. RESULTS: By combining RNAi-depletion experiments with in vitro biochemical assays, we demonstrate that the human kinesin Kif18A is a motile microtubule depolymerase essential for chromosome congression in mammalian tissue culture cells. We show that in vitro Kif18A is a slow plus-end-directed kinesin that possesses microtubule depolymerizing activity. Notably, Kif18A like its yeast ortholog Kip3p depolymerizes longer microtubules more quickly than shorter ones. In vivo, Kif18A accumulates in mitosis where it localizes close to the plus ends of kinetochore microtubules. The depletion of Kif18A induces aberrantly long mitotic spindles and loss of tension across sister kinetochores, resulting in the activation of the Mad2-dependent spindle-assembly checkpoint. Live-cell microscopy studies revealed that in Kif18A-depleted cells, chromosomes move at reduced speed and completely fail to align at the spindle equator. CONCLUSIONS: These studies identify Kif18A as a dual-functional kinesin and a key component of chromosome congression in mammalian cells.

Pre-anaphase chromosome oscillations are regulated by the antagonistic activities of Cdk1 and PP1 on Kif18A.

Upon congression at the spindle equator, vertebrate chromosomes display oscillatory movements which typically decline as cells progress towards anaphase. Kinesin-8 Kif18A has been identified as a suppressor of chromosome movements, but how its activity is temporally regulated to dampen chromosome oscillations before anaphase onset remained mysterious. Here, we identify a regulatory network composed of cyclin-dependent kinase-1 (Cdk1) and protein phosphatase-1 (PP1) that antagonistically regulate Kif18A. Cdk1-mediated inhibitory phosphorylation of Kif18A promotes chromosome oscillations in early metaphase. PP1 induces metaphase plate thinning by directly dephosphorylating Kif18A. Chromosome attachment induces Cdk1 inactivation and kinetochore recruitment of PP1α/γ. Thus, we propose that chromosome biorientation mediates the alignment of chromosomes at the metaphase plate by tipping the balance in favour of dephosphorylated Kif18A capable of suppressing the oscillatory movements of chromosomes. Notably, interfering with chromosome oscillations severely impairs the fidelity of sister chromatid segregation demonstrating the importance of timely controlled chromosome dynamics for the maintenance of genome integrity.

An in vitro system to study nuclear envelope breakdown.

During mitosis in vertebrate cells, the nuclear compartment is completely disintegrated in the process of nuclear envelope breakdown (NEBD). NEBD comprises the disassembly of nuclear pore complexes, disintegration of the nuclear lamina, and the retraction of nuclear membranes into the endoplasmic reticulum. Deciphering of the mechanisms that underlie these dynamic changes requires the identification of the involved molecular components and appropriate experimental tools to define their mode of action. Here, we describe an in vitro, imaging-based experimental system, which recapitulates NEBD. In our assay, we induce NEBD on nuclei of semi-permeabilized HeLa cells expressing fluorescently tagged nuclear envelope (NE) marker proteins by addition of mitotic cell extract that is supplemented with fluorescently labeled dextran. Time-lapse confocal microscopy is used to monitor the fate of the selected NE marker protein, and loss of the NE permeability barrier is deduced by influx of the fluorescent dextran into the nucleus. This in vitro system provides a powerful tool to follow NEBD and to characterize factors required for the reorganization of the NE during mitosis.

A non-motor microtubule binding site is essential for the high processivity and mitotic function of kinesin-8 Kif18A.

BACKGROUND: Members of the kinesin-8 subfamily are plus end-directed molecular motors that accumulate at the plus-ends of kinetochore-microtubules (kt-MTs) where they regulate MT dynamics. Loss of vertebrate kinesin-8 function induces hyperstable MTs and elongated mitotic spindles accompanied by severe chromosome congression defects. It has been reported that the motility of human kinesin-8, Kif18A, is required for its accumulation at the plus tips of kt-MTs. METHODOLOGY/FINDINGS: Here, we investigate how Kif18A localizes to the plus-ends of kt-MTs. We find that Kif18A lacking its C-terminus does not accumulate on the tips of kt-MTs and fails to fulfill its mitotic function. In vitro studies reveal that Kif18A possesses a non-motor MT binding site located within its C-proximal 121 residues. Using single molecule measurements we find that Kif18A is a highly processive motor and, furthermore, that the C-terminal tail is essential for the high processivity of Kif18A. CONCLUSION/SIGNIFICANCE: These results show that Kif18A like its yeast orthologue is a highly processive motor. The ability of Kif18A to walk on MTs for a long distance without dissociating depends on a non-motor MT binding site located at the C-terminus of Kif18A. This C-proximal tail of Kif18A is essential for its plus-end accumulation and mitotic function. These findings advance our understanding of how Kif18A accumulates at the tips of kt-MTs to fulfill its function in mitosis.